The renin-angiotensin system (RAS) plays a key role in regulating cardiovascular homeostasis and electrolyte/fluid balance in normotensive and hypertensive subjects.1-6 Angiotensin II (AII), an octapeptide that is formed within the RAS from angiotensin I by angiotensin-converting enzyme (ACE), is one of the most powerful vasoconstrictors known. AII was also found to be a growth factor implicated in cardiac, vascular hypertrophy and ventricular remodeling following myocardial infarction. Consequently, the RAS has been a prime target for the therapy of cardiovascular diseases. Reducing the levels of AII by inhibition of ACE is a good approach for treating hypertension, confirmed by the success of ACE inhibitors as antihypertensives. However, due to the fact that ACE inhibitors may increase the levels of bradykinin and cause side effects such as dry cough and angioedema (as do some AII antagonists), drugs that can antagonize AII at its receptor sites have been considered a more specific approach to blockade of the RAS. Although peptide analogues of AII such as sarilesin and sarmesin inhibit the action of AII by competitively binding to the receptor, their application as clinical agents is limited due to their short duration of action, poor bioavailability and partial agonist activity. However, these AII type I and type II30 antagonists have been valuable tools in our hands for identifying pharmacophoric groups and for the design of AII non-peptide mimetics. The discovery by DuPont of the first potent and orally active non-peptide AII antagonist Losartan has stimulated extensive research interest in this area. Several patents and publications have appeared over the past few years describing new AII receptor antagonists including Candesartan, Irbesartan, Valsartan, Telmisartan, Tasosartan and Eprosartan, which have been proven safe and effective in the treatment of hypertension and other cardiovascular disorders.
Our work has been focused in recent years on the study of the conformational analysis of the peptide hormone AII, the competitive antagonist sarmesin as well as other cyclic peptide derivatives. Comparative studies of sarmesin with AT1 receptor antagonists possessing different anti-hypertensive efficacies gave clues about the relation between conformation and bioactivity.
(A) Angiotensin I Receptor Antagonists: the Role of Negative Charges and Alkyl or Ester/Carboxyl Groups at Position 5 of Imidazole
Angiotensin II is an octapeptide hormone (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) which is a powerful arterial vasoconstrictor that exerts its action by interacting with specific receptors present on cell membranes. Blockade of the actions of angiotensin II using angiotensin receptor antagonists is useful for the treatment of hypertension and congestive heart failure and other cardiovascular and related diseases such as diabetic nephropathy. Pioneering work based on modifications of the peptide structure of ANG II led to potent modified peptides (Sarilesin, Saralasin, Sarmesin) that showed potent and selective in vitro ANG II receptor antagonism. However, the action of such agents in vivo was severely diminished by their rapid metabolism to inactive compounds. Thus, the search was on to identify and develop a non-peptide ANG II receptor antagonist that was both resistant to the metabolic deactivation of peptide antagonists and selective for the ANG II receptor.
In 1982, Takeda (Japan) filed a patent application disclosing the discovery of non-peptide ANG II receptor antagonists. The activity of these initial compounds was low but showed good selectivity. In subsequent years, much detailed knowledge was obtained through work on modified peptides, and DuPont engaged in extensive studies to exploit Takeda's early lead. These efforts were rewarded with the development of DuP753 (Losartan), which is now used to treat various hypertensive conditions. The antihypertensive activity of Losartan is largely due to a long-acting metabolite (EXP 3174) which is produced in vivo as a result of the conversion of hydroxymethyl to carboxylate, providing a negative charge required for affinity. Likewise, valsartan (CGP 48933) is a potent angiotensin receptor antagonist containing a carboxylate group analogous to that in EXP 3174. Indeed, a common feature of EXP 3174, CGP 48933 and another angiotensin mimetic SK 108566, is the presence of two acid groups spaced at similar distances on various aromatic templates.

Many other companies have sought to develop their own angiotensin mimetics in a bid to compete for a share in the huge worldwide market for antihypertensive drugs. From these molecules seven antagonists are in the market. Valsartan is the second molecule after Losartan to reach the market, while Irbersartan, Eprosartan, Candesartan, Telmisartan, Tasosartan and Olmesartan 10 followed the pipeline.

Inhibitors of Angiotensin in the Market
Reorientation of the imidazole ring of Losartan with relocation of butyl (aliphatic) and CH2OH-groups provided novel compounds useful in treating hypertension in anesthetized rats and rabbits. Based on a survey of four of the possible five orientations of the imidazole ring of Losartan, it was observed that compounds in which the biphenyl moiety is attached to an imidazole N atom, rather than one of the C atoms of the heterocyclic ring, have the highest activity. With this knowledge, we developed a series of compounds in an attempt to attain the high biological activities observed for Losartan itself. Transposition of the substituents at 2 and 5-positions of the imidazole ring of Losartan has provided compounds with significant activities in vitro when examined in the rat isolated uterus assay and anesthetized rabbits. Further protection of tetrazole by protecting groups as Trt, Cl-Trt, Benzyl and derivatives increased lipophilicity and furthermore the duration of the activity in anesthetized rats and rabbits. Lipophilic compounds were effective to reduce blood pressure in animal models by transdermal delivery.
With the exception of Eprosartan, the majority of the above non-peptide antagonists are based on modifications to one or more fragments of Losartan. Thus, there are a number of structural similarities between the compounds on the market.    (a) they generally have a biphenyl scaffold;    (b) the first phenyl (“spacer”) is connected with a nitrogen heterocycle and the second phenyl (“terminal”) with an acidic group such as carboxylic group, tetrazole, sulfonylurea, triflamide or substituted sulfonamide;    (c) most heterocycle rings attached to biphenyl tetrazole (BPT) possess adjacent groups like carboxy groups, basic nitrogen moieties, lactam oxygens that allow hydrogen-bonding to the corresponding acceptor;    (d) all molecules possess an alkyl chain attached to heterocycle; these alkyl chains are believed to fit a lipophilic pocket in the AT1 receptor.(B) Skin Delivery of Drugs
The most widely used routes for the administration of a drug to patients is either by providing it as a pill by mouth (per os) or by directly delivering it as an intramuscular or intravenous injection. However, lipophilic molecules can be delivered through the skin, as has been the case in various dermatologic remedies. In fact, besides the usual nutritive vessels, such as arteries, capillaries and veins, skin contains an extensive subcutaneous venous plexus, which can hold large quantities of blood. By delivering medications through the skin directly into the veins, one can bypass portal circulation and eliminate first pass metabolism in the liver, therefore eliminating side effects from active metabolites.
In addition to dermatologic preparations, other systemic drugs have been developed as transdermal patches. For example, the first transdermal nitroglycerine patch obtained FDA approval in 1981 and gained wide acceptance for its convenience. More recently, the transdermal formulation of oxybutynin was approved by FDA in 2003. As shown in the following Table, at least 8 different categories of drugs are currently manufactured as transdermal patches and they are used for slow, constant and prolonged release of their respective medication through the skin.
MedicationIndicationNitroglycerineChest pain due to coronary artery diseaseClonidineHigh blood pressure, opioid withdrawalEstrogens/ProgesteroneContraceptionTestosteroneMale hypogonadismNicotineSmoking CessationScopolamineVertigo, motion sicknessOxybutininBladder overactivityFentanylCancer pain
Transdermal nitroglycerine patches have been used for many years to prevent angina pectoris (exercise-induced chest pain due to coronary artery disease). Those patches either have a polymer matrix or a silicone gel impregnated with nitroglycerine. A semipermeable membrane between the drug reservoir and the skin results in a constant delivery of nitroglycerine. The onset of action is within 30 minutes, and the peak effect is seen in 60-180 minutes, with a duration of action of 8-14 hours.
Catapres-TTS is a multilayered film, 0.2 mm thick, containing clonidine as the active agent. To date, this formulation is the only one providing antihypertensive medication through the skin. In addition, clonidine patches have been used to decrease withdrawal symptoms in patients taking opioids. The amount of drug delivered is directly proportional to the size of the patch used. There are four layers: (a) a backing layer of polyester film, (b) a drug reservoir of clonidine, mineral oil, polyisobutylene and colloid silicon dioxide, (c) a microporous polypropylene membrane that controls the rate of delivery of clonidine and (d) an adhesive formulation. The patch is programmed to release clonidine at constant rate for 7 days. Allergic contact sensitisation is observed in 5% of patients.

Ortho Evra developed a transdermal contraceptive patch for providing hormonal contraception. Each patch delivers 20 g of ethinyl estradiol and 150 g of norelgestonin daily. The patch is changed once a week. In randomized studies the contraceptive efficacy of the patch was similar to that of oral contraceptives, but the compliance was significantly better with the patch. Discontinuation of the patch due to reaction at the site of application occurred in 1.9% of the women.
Transdermal delivery of testosterone first became available in 1994, as a scrotal patch. Since then more testosterone patches have been developed for the bare skin. Their major advantage is maintenance of stable serum testosterone concentration in the majority of patients. Testosterone can be also delivered through the skin a hydroalcoholic gel preparation approved by FDA in 2000. The only indication for their use is as replacement therapy in male hypogonadism.
Transdermal nicotine systems can deliver nicotine at several dosages and have been used for smoking cessation. Randomized studies have revealed that nicotine patches at higher dosage range associated with behavioral intervention may double the quitting rates when compared with behavioral intervention alone.
Scopolamine patches have been used to prevent motion sickness, as preoperative medication or to decrease excessive motility of the genitourinary or gastrointestinal tract. Scopolamine patches should be applied behind the ear 2-3 hours before anticipated need. They can deliver up to 1 mg of scopolamine over 3 days.
Oxybutynin relaxes bladder smooth muscles by blocking the muscarinic receptors and has been used in patients with bladder overactivity (urge urinary incontinence, frequency, nocturia). A transdermal formulation of oxybutynin was approved by FDA in 2003. Randomized studies have shown that transdermal oxybutynin was as effective as per os formulations, but direct delivery of the drug into the skin veins was associated with a lower incidence of dry mouth. Local pruritus was seen in 14% of patients using oxybutynin patches versus in 4% with placebo.
Fentanyl is the only opioid prepared for transdermal use in patients with cancer pain. The onset of analgesia is 12-14 hours from patch application and analgesia continues for 16-24 hours after removal of the patch.
Non-Peptide Mimetics of Angiotensin: from Peptides to Cyclic and Non-Peptide Mimetics of Angiotensin
The methodology to transfer angiotensin to its non-peptide mimetic antagonist includes the following steps: a) The Peptide (The tool), b) The Peptide Model (The Ligand-Receptor Interaction), c) The Cyclic Peptide (the Drug Lead) and d) the Non-Peptide Mimetic (the Drug).
The methodology to transfer angiotensin to its non-peptide mimetic antagonist is shown graphically in FIG. 1.
a) Peptide (the Tool)

Angiotensin II is consisting of eight aminoacids. Structure-activity studies have revealed the importance of residues Arg2, Tyr4, His6, Phe8 and the C-terminal carboxylate for activity. Peptide antagonists such as Sarilesin and Sarmesin cannot be used as drugs against hypertension due to metabolic degradation. Therefore the peptide hormone angiotensin can be used only as a tool to design non-peptide mimetics as drugs.
b) Peptide Model (the Ligand-Receptor Interaction)
Conformational Model
In 1994 a model was developed of angiotensin II which involves an aromatic ring cluster and consequently a charge relay system formed from the triad of aminoacids Tyr4-His6-Phe8. These three aminoacids are a strict requirement for angiotensin II to exert its agonist activity. Comparative nuclear magnetic resonance studies of the backbone structure between peptide agonists and antagonists have shown that agonists display ring clustering and form a change relay system. Such clustering is also present in the competitive antagonist Tyr(Me)4 ANG II (Sarmesin) which lacks the potential of the charge relay system and the form of the tyrosinate anion which is a strict requirement for agonist activity in the proposed model. In addition, the proposed conformation of ANG II overlays the recently discovered nonpeptide ANG II receptor antagonist EXP-3 174 and its analogs when molecular modeling techniques and superimposition studies are applied. Finally, the ring cluster conformation is supported by the design and synthesis of a novel constrained ANG II cyclic analogue [Sar1, Lys3, Glu5] ANG II, which possesses agonist activity when tested in the rat uterus assay and in anesthesized rabbits. This potent cyclic analog was designed with the integrity of the ring cluster as a major molecular feature. Based on structure activity relationships which demand the presence of Phe, Tyr and His for ANG II to possess biological activity, it can be inferred that the ability to form a ring cluster, and consequently a charge relay system, may be the key stereoelectronic molecular features of ANG II for exerting biological activity.
Theoretical calculations improved further the model and the revised one includes electrostatic interactions between Asp1-Arg2 and Arg2-Tyr4.
c) Cyclic Peptide (the Drug Lead)
Of all cyclic structures shown, only Cyclo (3-5) [Sar1, Glu3, Lys5] ANG II retains the ring cluster, i.e. the Phe, Tyr, His side chains on the same plane show biological activity. Cyclo (3-5) [Sar1, Glu3, Lys5, Ile8] ANG II, as expected according to the model, shows antagonist activity. All others are inactive as the integrity of cluster is lost.
